Designed reduction of Streptococcus pneumoniae pathogenicity via synthetic changes in virulence factor codon-pair bias.

In this study, we used a previously described method of controlling gene expression with computer-based gene design and de novo DNA synthesis to attenuate the virulence of Streptococcus pneumoniae. We produced 2 S. pneumoniae serotype 3 (SP3) strains in which the pneumolysin gene (ply) was recoded with underrepresented codon pairs while retaining its amino acid sequence and determined their ply expression and pneumolysin production in vitro and their virulence in a mouse pulmonary infection model. Expression of ply and production of pneumolysin of the recoded SP3 strains were decreased, and the recoded SP3 strains were less virulent in mice than the wild-type SP3 strain or a Δply SP3 strain. Further studies showed that the least virulent recoded strain induced a markedly reduced inflammatory response in the lungs compared with the wild-type or Δply strain. These findings suggest that reducing pneumococcal virulence gene expression by altering codon-pair bias could hold promise for rational design of live-attenuated pneumococcal vaccines.

[1]  D. Metzger,et al.  Interleukin-12 Promotes Gamma Interferon-Dependent Neutrophil Recruitment in the Lung and Improves Protection against Respiratory Streptococcus pneumoniae Infection , 2007, Infection and Immunity.

[2]  Su-Ting T. Li,et al.  Li S-TT and Tancredi DJ. Empyema Hospitalizations Increased in US Children Despite Pneumococcal Conjugate Vaccine. Pediatrics. 2010;125(1):26–33 , 2010, Pediatrics.

[3]  L. Pirofski,et al.  Efficacy of Opsonic and Nonopsonic Serotype 3 Pneumococcal Capsular Polysaccharide-Specific Monoclonal Antibodies against Intranasal Challenge with Streptococcus pneumoniae in Mice , 2009, Infection and Immunity.

[4]  M. Bergeron,et al.  Ceftriaxone pharmacokinetics in interleukin-10-treated murine pneumococcal pneumonia. , 2005, The Journal of antimicrobial chemotherapy.

[5]  P. Andrew,et al.  CD4-T-Lymphocyte Interactions with Pneumolysin and Pneumococci Suggest a Crucial Protective Role in the Host Response to Pneumococcal Infection , 2004, Infection and Immunity.

[6]  T. Ikemura.  Codon usage and tRNA content in unicellular and multicellular organisms. , 1985, Molecular biology and evolution.

[7]  G. Pier,et al.  Antibody-mediated enhancement of community-acquired methicillin-resistant Staphylococcus aureus infection , 2010, Proceedings of the National Academy of Sciences.

[8]  J. R. de los Toyos,et al.  A SPECIFIC AND ULTRASENSITIVE CHEMILUMINESCENT SANDWICH ELISA TEST FOR THE DETECTION AND QUANTITATION OF PNEUMOLYSIN , 2001, Journal of immunoassay & immunochemistry.

[9]  D. Briles,et al.  Differences in virulence for mice among Streptococcus pneumoniae strains of capsular types 2, 3, 4, 5, and 6 are not attributable to differences in pneumolysin production , 1997, Infection and immunity.

[10]  J. R. Coleman,et al.  Virus Attenuation by Genome-Scale Changes in Codon Pair Bias , 2008, Science.

[11]  O. Avery,et al.  STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES , 1944, The Journal of experimental medicine.

[12]  R. Möllby,et al.  Production and purification of Streptococcus pneumoniae hemolysin (pneumolysin) , 1987, Journal of clinical microbiology.

[13]  J. Weiser,et al.  Live Attenuated Streptococcus pneumoniae Strains Induce Serotype-Independent Mucosal and Systemic Protection in Mice , 2007, Infection and Immunity.

[14]  Maido Remm,et al.  Preferred and avoided codon pairs in three domains of life , 2008, BMC Genomics.

[15]  D. Briles,et al.  Reduced virulence of a defined pneumolysin-negative mutant of Streptococcus pneumoniae , 1989, Infection and immunity.

[16]  P. Andrew,et al.  Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1 beta by human mononuclear phagocytes , 1994, Infection and immunity.

[17]  S. Normark,et al.  Streptococcus pneumoniae evades human dendritic cell surveillance by pneumolysin expression , 2009, EMBO molecular medicine.

[18]  C. Dye,et al.  Estimates of world-wide distribution of child deaths from acute respiratory infections. , 2002, The Lancet. Infectious diseases.

[19]  R. Dagan,et al.  Streptococcus pneumoniae Serotype 3 among Costa Rican Children with Otitis Media: clinical, epidemiological characteristics and antimicrobial resistance patterns , 2009, BMC pediatrics.

[20]  J. Moreland,et al.  Neutrophil transendothelial migration in vitro to Streptococcus pneumoniae is pneumolysin dependent. , 2006, American journal of physiology. Lung cellular and molecular physiology.

[21]  Su-Ting T. Li,et al.  Empyema Hospitalizations Increased in US Children Despite Pneumococcal Conjugate Vaccine , 2010, Pediatrics.

[22]  A. Ferrante,et al.  Inhibition of human polymorphonuclear leukocyte respiratory burst, bactericidal activity, and migration by pneumolysin , 1983, Infection and immunity.

[23]  J. Gerberding,et al.  Direct and indirect effects of routine vaccination of children with 7-valent pneumococcal conjugate vaccine on incidence of invasive pneumococcal disease--United States, 1998-2003. , 2005, MMWR. Morbidity and mortality weekly report.

[24]  J. Tschopp,et al.  Pneumolysin Activates the NLRP3 Inflammasome and Promotes Proinflammatory Cytokines Independently of TLR4 , 2010, PLoS pathogens.

[25]  P. Andrew,et al.  The role of pneumolysin in pneumococcal pneumonia and meningitis , 2004, Clinical and experimental immunology.

[26]  D. Briles,et al.  Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 streptococcus pneumoniae , 1981, The Journal of experimental medicine.

[27]  M. Gouy,et al.  Codon usage in bacteria: correlation with gene expressivity. , 1982, Nucleic acids research.

[28]  L. Pirofski,et al.  Pneumococcal Capsular Polysaccharide Vaccine-Mediated Protection against Serotype 3 Streptococcus pneumoniae in Immunodeficient Mice , 2007, Infection and Immunity.

[29]  G. W. Hatfield,et al.  Nonrandom utilization of codon pairs in Escherichia coli. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[30]  W. Schaffner,et al.  Incidence of pneumococcal disease due to non-pneumococcal conjugate vaccine (PCV7) serotypes in the United States during the era of widespread PCV7 vaccination, 1998-2004. , 2007, The Journal of infectious diseases.

[31]  D. Briles,et al.  The potential for using protein vaccines to protect against otitis media caused by Streptococcus pneumoniae. , 2000, Vaccine.

[32]  J. Weiser,et al.  Neutrophil-Toxin Interactions Promote Antigen Delivery and Mucosal Clearance of Streptococcus pneumoniae1 , 2008, The Journal of Immunology.

[33]  B. Baban,et al.  B-lymphoid cells with attributes of dendritic cells regulate T cells via indoleamine 2,3-dioxygenase , 2010, Proceedings of the National Academy of Sciences.

[34]  L. Pirofski,et al.  Influence of Neutropenia on the Course of Serotype 8 Pneumococcal Pneumonia in Mice , 2007, Infection and Immunity.

[35]  Steven Skiena,et al.  Live Attenuated Influenza Vaccines by Computer-Aided Rational Design , 2010, Nature Biotechnology.

[36]  CD8+ Cells Enhance Resistance to Pulmonary Serotype 3 Streptococcus pneumoniae Infection in Mice , 2011, The Journal of Immunology.

[37]  P. Andrew,et al.  Human Lung Mast Cells Mediate Pneumococcal Cell Death in Response to Activation by Pneumolysin , 2010, The Journal of Immunology.

[38]  C. Byington,et al.  Molecular Epidemiology of Pediatric Pneumococcal Empyema from 2001 to 2007 in Utah , 2009, Journal of Clinical Microbiology.

[39]  G. Deepe,et al.  The CCL7-CCL2-CCR2 Axis Regulates IL-4 Production in Lungs and Fungal Immunity1 , 2009, The Journal of Immunology.

[40]  J. R. de los Toyos,et al.  Immunodetection of pneumolysin in human urine by ELISA. , 2003, Journal of microbiological methods.

[41]  J. Paton,et al.  Effect of immunization with pneumolysin on survival time of mice challenged with Streptococcus pneumoniae , 1983, Infection and immunity.

[42]  Thomas B. Clarke,et al.  Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice. , 2009, The Journal of clinical investigation.

[43]  E. Tuomanen,et al.  Inhibition of T Cells Provides Protection against Early Invasive Pneumococcal Disease , 2010, Infection and Immunity.

[44]  G. Ehrlich,et al.  Characterization, Distribution, and Expression of Novel Genes among Eight Clinical Isolates of Streptococcuspneumoniae , 2006, Infection and Immunity.

[45]  M. Lipsitch,et al.  Construction of Otherwise Isogenic Serotype 6B, 7F, 14, and 19F Capsular Variants of Streptococcus pneumoniae Strain TIGR4 , 2003, Applied and Environmental Microbiology.

[46]  M. Lipsitch,et al.  Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[47]  J. Poolman,et al.  Pneumococcal serotype 3 otitis media, limited effect of polysaccharide conjugate immunisation and strain characteristics. , 2009, Vaccine.

[48]  A. Finn,et al.  Induction of CC and CXC chemokines in human antigen-presenting dendritic cells by the pneumococcal proteins pneumolysin and CbpA, and the role played by toll-like receptor 4, NF-kappaB, and mitogen-activated protein kinases. , 2008, The Journal of infectious diseases.

[49]  D. Briles,et al.  Development of a Vaccine against Invasive Pneumococcal Disease Based on Combinations of Virulence Proteins of Streptococcus pneumoniae , 2006, Infection and Immunity.

[50]  M. Fujimoto,et al.  A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T cell-dependent inflammatory responses. , 2008, Immunity.

[51]  M. Lipsitch,et al.  Interleukin-17A Mediates Acquired Immunity to Pneumococcal Colonization , 2008, PLoS pathogens.